Rand K H, Houck H
Department of Pathology, University of Florida, Gainesville.
Mol Cell Probes. 1990 Dec;4(6):445-50. doi: 10.1016/0890-8508(90)90003-i.
The polymerase chain reaction (PCR) was carried out with the highly conserved E. coli ribosomal RNA gene sequences 1376-1395 and 1521-1540. Using these primers and reaction conditions specified by the manufacturer(s), a 165 bp fragment was synthesized using Taq polymerase from three different sources in the absence of any added template. Restriction enzyme analysis suggests the source of this bacterial DNA is neither E. coli nor Thermus aquaticus. A variety of different methods to eliminate it such as treatment with DNase, restriction enzyme digestion, and CsCl2 density gradient centrifugation were unsuccessful. Since the bacteria in which the Taq polymerase is produced are not the source of the DNA, some step(s) in the purification or reagents added to the enzyme must be involved. Thus it is likely other biological products are similarly contaminated. Although the problem is easily dealt with by running a no-template control and choosing other primers if a problem exists, it is important to recognize the potential for a false-positive result.
采用高度保守的大肠杆菌核糖体RNA基因序列1376 - 1395和1521 - 1540进行聚合酶链反应（PCR）。使用这些引物以及制造商指定的反应条件，在未添加任何模板的情况下，利用来自三种不同来源的Taq聚合酶合成了一个165 bp的片段。限制性内切酶分析表明，这种细菌DNA的来源既不是大肠杆菌也不是嗜热水生栖热菌。多种不同的消除方法，如用DNase处理、限制性内切酶消化和CsCl2密度梯度离心均未成功。由于生产Taq聚合酶的细菌不是DNA的来源，那么酶的纯化过程中的某些步骤或添加到酶中的试剂必定与此有关。因此，其他生物制品可能同样受到污染。尽管通过进行无模板对照以及在存在问题时选择其他引物，这个问题很容易解决，但认识到出现假阳性结果的可能性很重要。
